DESCRIPTION: (Applicant's Description) Many signaling peripheral proteins, including protein kinases C (PKC), are translocated to different cell membranes upon cell activation. Recent studies have shown that the 41embrane targeting and activati6n of diverse peripheral proteins is mediated by a small number of membrane targeting domains, including Cl and C2 domains. During the past granting period, we have studied the in vitro membrane binding and activation mechanisms of PKC isoforms, which have led to a working model that explains much of the temporal and spatial sequences of its membrane targeting and activation. The primary objective in the next granting period is to elucidate the mechanisms of both the in vitro and cellular membrane targeting and activation of different PKC isoforms containing Cl and C2 domains, with an emphasis on understanding how these domains interact with PKC activators and with each other to achieve specific membrane targeting and exquisite PKC regulation. A long-term objective is to apply the principles learned from these studies to the development of therapeutic agents that can specifically modulate the targeting and activation of signaling proteins. Specific aims for the next granting period are as follows, (1) further elucidation of the in vitro membrane binding and activation mechanisms of conventional PKCs, including PKC-alpha and PKC-gamma, (2) determination of the in vitro membrane binding and activation mechanisms of novel PKCs, including PKC-delta and PKC-epsilon, and (3) determination of the cellular membrane translocation and activation mechanisms of these PKCs. The principal methodologies to be used include: (1) the site-directed mutagenesis and the overexpression 0: PKC isoforms, (2) membrane-binding analysis of PKC isoforms with various model membranes developed in this laboratory and the surface plasmon resonance analysis that allows direct measurement of membrane association and dissociation rates, and (3) cell transfection of PKC isoforms and their mutants that are tagget with green fluorescent protein, followed by two-photon microscopic analysis.